Amphotericin B is an effective antifungal polyene macrolide antibiotic. This drug acts at the membrane level, ultimately inducing cation leakage and lysis of the target cell. It has also been shown to inhibit the infection of cultured cells by the human immunodeficiency virus (HIV) and may enhance the effectiveness of other anti-AIDS drugs. In addition, Amphotericin B has been studied as a simple model system for transmembrane ion channels. There are still many aspects of the molecular mechanism of activity of this and other structurally related drugs which are unclear. There are still many aspects of the molecular mechanism of activity of this and other structurally related drugs which are unclear. In this study, we hope to perfect new quantitative techniques for investigating these and other membrane-active antibiotics. Stopped-flow kinetic fluorescence probe techniques will be developed for detecting antibiotic induced ion currents across membrane vesicles. This study will focus mainly on three probes: 1) a DANSYL-triphenyl phosphonium, 2) 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), and 3) pyranine. These probes can detect currents of any net ion flow, chloride, or H+/OH- influx or efflux, respectively, across synthetic or natural membrane vesicles. Simple thermodynamic models relating the time-dependent redistribution of probe 1) to electrogenic ion currents will be development such that estimated transmembrane voltage versus fluorescence calibration curves are not necessary. To achieve this, we will relate probe binding in the absence of potential to fluorescence intensity in a given system. Changes in transmembrane voltage (currents) may then be related to time-dependent probe redistribution and binding. Probes 2) and 3), on the other hand, respond directly and rapidly to effluxes or influxes of the appropriate species without redistribution. Chemical gradients of various salts and pH values will be created with the variable ratio syringes of the stopped-flow spectrometer and the resolution in the millisecond range should be possible. Combinations of experiments with these probes should provide a clearer picture of the mechanisms of polyene macrolide antibiotic activity. These methods will also be generally applicable to a wide variety of problems involving biological transmembrane channels.